基于核酸碱基猝灭荧光团的核酸适配体传感器检测赭曲霉毒素A  

Aptasensor Based on Nucleic Acid Base Quenching Fluorophore for Detection of Ochratoxin A

作　　者：曲瑶[1] 张亚旗 肖光[1] 杨成[1] QU Yao;ZHANG Ya-Qi;XIAO Guang;YANG Cheng(State Key Laboratory of Fine Chemicals,School of Chemistry,College of Chemical Engineering,Dalian University of Technology,Dalian 116024,China)

机构地区：[1]大连理工大学化工学院化学系,精细化工国家重点实验室,大连116024

出　　处：《分析化学》2020年第10期1409-1415,共7页Chinese Journal of Analytical Chemistry

基　　金：中央高校基本科研业务费专项资金(No.DUT15RC(3)064);辽宁省博士科研启动基金(No.201501167);电分析化学国家重点实验室开放课题(No.SKLEAC201604)资助。

摘　　要：赭曲霉毒素A(OTA)是农产品中常见的霉菌毒素,对人和动物具有较强的毒性,以及致畸、致癌、致突变作用,因此,建立简单、快速、低成本、高灵敏的OTA检测方法是即时农产品质量监测和保障消费者安全的有效手段。本研究以鸟嘌呤碱基为猝灭剂,以单标记荧光素(FAM)的寡聚核酸为探针,基于核酸适配体构建了生物传感器,用于检测葡萄酒中的OTA。鸟嘌呤是具有多个给电子基团的稠杂环化合物,电子密度较大,在核酸碱基中氧化电位最低,最容易被氧化,作为电子供体可通过光诱导电子转移过程猝灭荧光基团。OTA不存在时,标记有FAM的寡聚核酸探针与OTA核酸适配体杂交,FAM靠近鸟嘌呤荧光被猝灭;样品中存在OTA时,其与核酸适配体特异性结合,形成四链体结构,抑制寡聚核酸探针与适配体结合,FAM的荧光得以恢复,通过FAM荧光强度的恢复率实现对OTA的检测。本方法检测OTA的线性范围为0.67~7.80 nmol/L,检出限为0.67 nmol/L(0.27μg/kg,S/N=3)。实际红酒样品中OTA的加标回收率为92.4%~100.9%。与纳米金、单壁碳纳米管、氧化石墨烯等纳米材料为猝灭剂的方法相比,本方法具有检测成本低、选择性好、检出限低等优点。Ochratoxin A(OTA) is one kind of mycotoxin, which is toxic and widely distributed. To avoid the poison of OTA, it is necessary to develop a rapid, low-cost and highly sensitive detection method to evaluate the concentration of OTA in the food. Here, an aptamer biosensor was developed to detect OTA in wine using single labeled oligonucleotide with fluorescein(FAM) as probe and guanine in the aptamer as quencher. Guanine was a heterocyclic compound that had a high electron density and the lowest oxidation potential in nucleic acid bases. Guanine was easily to be oxidized. As an electron donor, the fluorophore could be quenched by guanine through the process of photoinduced electron transfer(PET). When the probe labeled with FAM hybridized with the OTA aptamer, the fluorescence of FAM was quenched by the guanine in OTA aptamer. In the presence of OTA, due to the specific interaction between OTA and aptamer, OTA induced aptamer folding to G-quadruplexes, which inhibited the binding of probe with aptamer, and thus led to the fluorescence intensity recovery. The concentration of OTA could be determined by the recovery ratio of FAM fluorescence intensity. The linear range for detection of OTA by this aptasenor was 0.67-7.80 nmol/L, the linear calibration equation was R(%) =-0.02+1.76COTA (nmol/L), and LOD was 0.67 nmol/L(0.27 g/kg, S/N=3).The recoveries of OTA in red wine sample were in the range of 92.4%-100.9%. Compared with sensing strategies based on gold nanoparticles, single-walled nanotubes and graphene oxide, this method had many advantages such as low cost, good selectivity and high sensitivity.

关 键 词：核酸碱基 荧光猝灭 核酸适配体 单标记荧光团 赭曲霉毒素A 

分 类 号：O657.3[理学—分析化学] TS207.5[理学—化学]